DESCRIPTION OF THE OVERALL U19 APPLICATION (provided by applicant): This proposal will use human gastrointestinal organoids as a model system. We will integrate state-of-the-art methodologies, with collaborative and synergistic research approaches to the study of gastrointestinal diseases. We will develop in vitro gastrointestinal models that replicate the biological structures of the human intestinal tract. These tissues will recapitulate human physiology and disease pathology, and incorporate components critical to disease and human host response. Our focus will span the pipeline from basic research (pathogenesis) to product development. For gastrointestinal pathogens that have no relevant animal models, another long-term goal is to develop model systems that can be used in IND-enabling studies. We will advance the field through the following Specific Aims: Aim 1. Title: Mechanisms of H. pylori-host infection using human gastric organoids. Research Project 1 will use the newly developed gastric organoid system to study Helicobacter pylori. The proposed studies have the potential to advance our understanding of the mechanisms by which H. pylori alters gastric stem cell fate, and advance our understanding of the role of H. pylori as a carcinogen. Aim 2. Title: Human Intestinal Organoids to Study Toxigenic Bacterial Pathogens. Research Project II will use human intestinal organoids as a novel model system to study two toxigenic enteric pathogens, Clostridium difficile and Shiga toxin (Stx) producing Escherichia coli (STEC). The proposed studies have the potential to advance our understanding of the pathogenic processes of C. difficile and STEC and create model systems for the evaluation of potential therapeutics. Aim 3. Title: Human gastroids and colonoids as 4D models of gastrointestinal infection. Research Project III will develop ex vivo models to determine age-dependent and temporal GI epithelial responses to: a) H. pylori infection in human gastric organoids (HGOs) and gastroids and b) Clostridium difficile (CD) and Shiga toxin-producing Escherichia coli (STEC) infection and their major toxins in human colonic organoids (HCOs) and colonoids. Aim 4. Technology dissemination. We will advance and encourage educational and training activities to investigators currently affiliated with this proposal, investigators funded through other NAMSED awards, and interested members of the scientific community in general. Aim 5. Organoid Engineering Core. The human organoid core will develop reporter lines in which fluorescent proteins are expressed in specific lineages. shRNA knockdown and CRISPR targeted lines will give rise to human gastro-intestinal organoids lacking specific lineages, and tetracycline-regulated systems will be used to expand specific lineages.